WBB6/F₁-Kit^W/Kit^Wv (W/W^v) female mice (8–12 wk old) and their female congenic littermates, WBB6/F₁-Kit⁺/Kit⁺ (F₁^+/+), were obtained from The Jackson Laboratory. Both of these groups result from the cross of WB/ReJ-Kit^W/+ × C57BL/6-Kit^Wv/+ mice. Animal care was provided according to protocols approved by the Institutional Animal Care and Use Committee of Emory University.

EAE induction was performed according to the protocol of Mendel et al. 25. In brief, 300 μg of myelin oligodendrocyte glycoprotein (MOG)_35–55 peptide MEVGWYRSPFSRVVHLYRNGK (Microchemical Facility, Emory University) was dissolved in 100 μl of PBS and emulsified in an equal volume of CFA (Difco Labs., Inc.) containing 5 mg/ml of Mycobacterium tuberculosis H37 RA (Difco Labs., Inc.). The emulsion (200 μl) was injected subcutaneously into the flank on days 0 and 7. Pertussis toxin, 500 ng in 500 μl of PBS (List Biological Labs.), was administered intravenously into each tail vein on days 0 and 2. Mice were scored daily according to the following clinical scoring system: 0, no clinical disease; 1, tail flaccidity; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb paralysis or loss of ability to right from supine; 5, death.

Bone marrow was harvested from both femurs of 6–8-wk-old wild-type F₁^+/+ female mice and cultured in complete RPMI media (15% heat-inactivated FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, and 50 μM 2-β-ME) containing 25% WEHI-3B supernatant as an IL-3 source 26. In contrast to some previously described methods for culturing bone marrow–derived mast cells (BMMCs; reference 272829), recombinant murine stem cell factor (12.5 ng/ml; R & D Systems, Inc.) was also added to the culture during the first 2 wk as described 3031. This addition consistently increased the viability of the cultured cells. BMMCs were used after a minimum of 4 wk in culture at >96% purity, as determined by flow cytometric analysis. At time of reconstitution, BMMCs (5 × 10⁶ in 300 μl) were injected intravenously into groups of five to seven W/W^v mice. Mice were housed for 10 wk before being subjected to EAE disease induction along with age-matched W/W^v and F₁^+/+ controls.

After animals were killed, brains, spinal columns, and other organs were removed and preserved in 10% neutral buffered formalin. Tissues were embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin or Giemsa.

BMMCs (10⁶ cells in 100 μl) were blocked with antibodies to the Fcγ receptors CD16 and CD32 (PharMingen). Cells were incubated with murine IgE (PharMingen) and then surface stained with directly conjugated mAbs to murine IgE (rat anti–mouse–FITC; PharMingen) and c-kit (c-kit–PE; PharMingen). Flow cytometric analyses for BMMC purity were carried out with the appropriate isotype controls. Cells double positive for c-kit and FcεRI were considered mast cells.

Antibody level analyses were performed by specific ELISA to detect anti-MOG activity. MOG (0.25 μg/well in 0.1 M NaHC0₃, pH 9.6) was adsorbed onto flat-bottomed microtiter plates overnight at 4°C. After a blocking step of PBS/0.3% Tween 20/5% nonfat dry milk, plates were incubated with 1:100 dilutions of mouse sera in PBS/0.3% Tween 20. Anti-MOG antibodies bound to the MOG-coated plate were detected using peroxidase-conjugated, affinity-purified IgG fractions of isotype-specific goat anti–mouse IgG, IgG1, IgG2a, IgG2b, or IgG3 (PharMingen) diluted 1:1,000 in PBS/0.3% Tween 20. Assays were developed with 3,3′,5,5′-tetramethylbenzidine peroxidase substrate (KPL), stopped with H₃PO₄ (1:20 dilution), and read at a wavelength of 450 nm on a microplate reader.

Statistical analyses were performed using GraphPad Prism (Software for Science). Group mean clinical scores were analyzed by paired t test for comparison of two groups. Repeated measures of analysis of variance (ANOVA), followed by the Bonferroni post-test, were used for comparison of the mean clinical scores of the three groups in the reconstitution experiments. Comparison of group incidence (number of animals with disease/n) was analyzed by Fisher's exact test. Survival curves (animals positive for disease) were plotted according to the method of Kaplan-Meier, and significance was calculated by the log-rank test. Mean high scores were compared by student's t test or ANOVA with Bonferroni post-test for comparison of two or three groups, respectively.

To directly evaluate the in vivo role of mast cells in acute EAE, mast cell–deficient WBB6/F₁-Kit^W/Kit^Wv (W/W^v) mice and their congenic wild-type WBB6/F₁-Kit⁺/Kit⁺ (F₁^+/+), littermates (H-2^bxj) were immunized with the encephalitogenic MOG_35–55 peptide. MOG can induce typical EAE disease in C57BL/6 mice and other H-2^b strains 25. MOG, which comprises only ∼0.05% of myelin proteins, elicits a major antibody response that has been correlated with disease severity and demyelination in both human disease and animal models of MS 323334. In three independent experiments, W/W^v mice developed significantly less severe disease than wild-type mice, as indicated by lower daily mean clinical scores (P < 0.0001; Fig. 1 A). In addition, mast cell–deficient animals also demonstrated a delayed onset and lower incidence of disease when compared with their wild-type counterparts (P < 0.0003; Fig. 1 B). Sham-immunized wild-type (n = 3) and mast cell–deficient animals (n = 4) that received pertussis toxin and adjuvant alone showed no clinical signs of disease (data not shown). The cumulative analyses of disease parameters are presented in Table .

In addition to the clinical changes observed, animals were also examined for histologic evidence of disease induction. Initially, we confirmed the presence of CNS mast cells in naive animals used in this model system. Using metachromatic staining, mast cells were identified in CNS samples of wild-type mice only, particularly in perivascular regions of the hippocampus, leptomeninges, habenula, and thalamus (Fig. 2A and Fig. B). Tissue samples from immunized wild-type and W/W^v mice were also examined for the presence of inflammatory lesions. Typical mononuclear infiltrates with perivascular cuffing were noted in the brains and spinal cords of diseased mice in both groups (Fig. 2C and Fig. D). No apparent differences between the two groups were observed in the composition or distribution of inflammatory infiltrates. Mast cells were not identified in the inflammatory lesions of wild-type mice, consistent with the previous findings of Ibrahim et al. 35 in which mast cells were found in lesions from chronic but not acute disease.

If mast cell deficiency alone accounts for the differences in EAE disease parameters observed in W/W^v mice, reconstitution of the mast cell population in these animals should restore disease incidence and severity to the level of wild-type animals. The development of functional mast cells is dependent on the interaction of stem cell factor (SCF) with its receptor, c-kit, expressed on bone marrow–derived pluripotent stem cells. The mast cell deficiency of W/W^v mice is due to mutations in c-kit that compromise its signaling function 3637. The mast cell population can be reconstituted in these animals by intravenous injection of c-kit⁺ bone marrow cells or in vitro–differentiated, bone marrow–derived mast cell precursors (BMMCs) from wild-type animals 42729383940414243. Mast cell numbers in the skin, respiratory tract, and gastrointestinal tissues of the W/W^v mice after transplantation with either bone marrow cells or BMMCs are comparable to those of wild-type animals by 10–12 wk after transplantation. Importantly, the phenotypic characteristics of these cells resemble the local, native populations of mast cells in normal mice 293839.

We performed mast cell reconstitution in 8–10-wk-old W/W^v recipients by intravenous injection of BMMCs (>96% purity, as determined by flow cytometry; Fig. 3) to repair the mast cell deficit. To assess the establishment of mast cells in these mice, animals were killed 14–16 wk after reconstitution, and major organs were examined for the presence and distribution of mast cells. Mast cells were observed in the gut, CNS, and bone marrow as well as other organs in distribution patterns consistent with those seen in wild-type mice (Fig. 4). As expected, no mast cells were detected in tissues obtained from W/W^v mice.

The selectivity of the mast cell reconstitution was confirmed by hematocrit (Hct) determination 27394041. W/W^v mice are anemic (Hct 38.0 ± 3.0%) compared with wild-type F₁^+/+ mice (Hct 51.5 ± 0.71%). Reconstituted W/W^v mice remain anemic (Hct 33.6 ± 3.1%) after BMMC transplantation, demonstrating that all hematologic deficits are not restored by this procedure.

10 wk after reconstitution, BMMC recipients as well as age-matched wild-type and W/W^v mice were subjected to the EAE disease induction protocol. As shown in Fig. 5 A, reestablishment of the mast cell population in W/W^v mice completely restored the ability of these animals to develop severe disease. When compared with wild-type mice, the mast cell–reconstituted animals showed a similar time of onset, daily mean clinical score, and disease incidence (Fig. 5). Inflammatory infiltrates in the brain and spinal cord were also similar (data not shown). In all disease parameters examined, significant differences existed between mast cell–deficient mice and those with intact mast cell compartments (Fig. 5 and Table ).

In the reconstitution experiments, it was noted that wild-type and W/W^v animals demonstrated higher mean clinical scores than those observed in younger animals of respective genotypes (Fig. 1 A). In addition, some individual W/W^v mice had clinical scores as high as those of the wild-type animals. The explanation for these observations is unclear, but they may be due to age-related differences in host sensitivity to pertussis toxin or peptide dose. These possibilities are presently being examined.

While it is formally possible that W/W^v mice have T cell deficits that could account for the differences in disease parameters demonstrated between wild-type and mast cell–deficient animals, we believe this is unlikely. Thymocytes are c-kit⁺, and the defect in c-kit carried by W/W^v mice could potentially hinder T cell development in these animals; however, previous characterizations of W/W^v mice revealed no such T cell deficits 4445. It has also been demonstrated that IL-7, which has many activities that overlap with SCF, can direct the development of normal T cells in c-kit–deficient mice 44. In addition, we evaluated MOG-specific proliferative responses, cytokine profiles, and antibody production in both groups. Splenocytes from MOG-immunized wild-type and W/W^v mice mounted equivalent proliferative responses and IFN-γ cytokine production in response to in vitro stimulation with MOG peptide (data not shown). No IL-4 was detected in these assays. Wild-type and W/W^v mice, as well as BMMC-reconstituted W/W^v animals, produced similar levels of MOG-specific IgG (Fig. 6). MOG-specific IgG1 and IgG2b subtypes were also detected in all three groups. Interestingly, the MOG-specific IgG1 levels of W/W^v and BMMC-reconstituted W/W^v mice were significantly higher (P < 0.05, ANOVA) than those of wild-type mice. The biological significance of this observation is unclear. However, it may indicate that c-kit signaling pathways play an as yet unidentified role in B cell isotype switching. Alternatively, the kinetics of IgG1 antibody production may be altered in these mutant animals. Despite these differences in IgG1 levels, it is unlikely that this has a major effect on the development of EAE, because wild-type and BMMC-reconstituted mice exhibit similar disease courses. Also of note, total serum IgE was high in immunized animals within all groups, yet MOG-specific IgE was undetectable (data not shown). These results indicate that there are no global T or B cell deficits in W/W^v mice. Taken together with the demonstration that mast cell reconstitution with a virtually pure BMMC population restores disease susceptibility, these data support the hypothesis that it is the absence of mast cells in the W/W^v animals that predisposes them to delayed onset and less severe disease.